Polynucleotide and protein involved in synaptogenesis variants thereof and their therapeutic and diagnostic uses

ABSTRACT

The present invention provides a method of diagnosing autism linked to a mutation in the polynucleotide of SEQ ID NO: 12 or the polypeptide of SEQ ID NO: 14, or a propensity therefor, in a human, where the mutation results in altered synapse formation.

CONTEXT OF THE INVENTION

a) Field of the Invention

The present invention relates to the identification of a human geneencoding a protein involved in synapto-genesis and of its murineorthologs, the mutation of which is associated in humans with thedevelopment of neurological diseases and/or with a predisposition to thedevelopment of mental disorders or psychiatric diseases such as autismand Asperger syndrome.

The invention also relates to the diagnostic and therapeutic usesassociated with identifying the gene and its mutations.

The invention also relates to the diagnostic and therapeutic usesassociated with identifying the involvement of a defect in a protein ofthe neuroligin family in the development of mental disorders orpsychiatric diseases such as autism and Asperger syndrome.

b) Brief Description of the Prior Art

Autism is a disease which affects around one child in 1000 and mainlyboys (from 4 to 23 boys to one girl according to the selected clinicalcriteria). The clinical symptoms of autism are described in theDSM-IV-TR™ manual (Diagnostic and statistical manual of mentaldisorders, 2000, pages 70-75).

The molecular bases of autism are currently unknown. Studies havealready suggested the existence of a genetic component in autism. Basedon linkage analysis results, Philippe et al. (Human Molecular Genetics,1999, 8:805-812) describes 11 chromosomal regions which may be involvedin the development of autism, among which is a region of chromosome Xp.In addition, Thomas et al. (Hum. Genet., 1999, 104:43-48) describesdeletions of the short arm of the X chromosome in patients sufferingfrom autism, and Milunsky et al. (Clin. Genet., 1999, 55:455-460)describes deletions in Xp22 in patients suffering from schizophrenia andsuggests, more precisely, the involvement of a deletion in Xp22.3 in thedevelopment of this psychiatric disease. However, neither the geneinvolved in the disease nor the nature of the mutation are mentioned.

The neuroligins (HNLs) are cell adhesion proteins which can trigger, bythemselves, synaptogenesis, i.e. the formation of synapses (Scheiffeleet al., Cell, 2000, 101:657-669). The neuroligins NL1, NL2 and NL3 wereoriginally cloned in rats (Ichtchenko et al., Cell., 1995, 81(3):435-43;Ichtchenko et al., J. Biol. Chem., 1996, 271(5):2676-82). Theneuroligins HNL1 and HNL2 are located on autosomes (3q26 and 17p13).These genes are targets for susceptibility to psychiatric diseases andseveral protein variations in HNL2 have been demonstrated by theinventors in autistic patients (R734H, G754R, A755V). A protein HNL4X(human neuroligin-4) has been described by Bollinger et al. (Biochem.J., 2001, 356:581-588), without any genomic description or relatedbiological function. In addition, the LOCUSLINK™ database of Sep. 13,2001 provides, under the accession numbers KIAA1260 and KIAA0951,incomplete sequences of a neuroligin gene, the function of which is alsounknown. All neuroligins have an extracellular domain homologous withacetylcholine esterase (ACHE). Neuroligins interact with β-neurexins, atthe level of this extracellular component (Ichtchenko et al., Cell.,1995, 81(3):435-43). This interaction can be modulated by ACHE itself(Grifman et al., Proc. Natl. Acad. Sci. USA, 1998, 95(23):13935-40). Atthe cytoplasmic level, neuroligins interact with several proteinscontaining PDZ domains (Irie et al., Sciences, 1997, 277(5331):1511-5;Hirao et al., J. Biol. Chem. 1998, 273(33), 21105-10; Kurschner et al.,Mol. Cell Neurosci., 1999, 11(3): 161-72; Bolliger et al., Biochem J.,2001, 356:581-8; Toyooka et al., J Neurochem., 2002, 83(4):797-806).Among these proteins, are the proteins of the DLG1-5 family (3q29,11q13, Xq13.1, 17p13.1 and 10q22.3), the S-SCAM protein (7q21.11), theproteins of the CIPP family (MPDZ, 9p23 and INADL, 1p31) and the CASKprotein (Xp11).

In view of the above, it is clear that knowledge of the molecular basesof autism, and most particularly of the gene involved in the disease, isgreatly desired in order to make it possible to develop noveltherapeutic approaches, novel medicinal products and diagnostic tests.

A need also exists concerning identification of the biological functionof neuroligins and also identification of the nucleic acid and proteinsequence of neuroligins, in particular that of HNL3 and HNL4X.

The present invention satisfies these needs and other needs, as will beapparent to those skilled in the art upon reading the presentdescription of the invention.

SUMMARY OF THE INVENTION

The present invention relates to the identification of human genes andof their murine ortholog encoding a protein involved in synaptogenesis,the mutation of which is associated, in humans, with the development ofneurological diseases and/or with a predisposition to the development ofmental disorders or psychiatric diseases such as autism and Aspergersyndrome.

More particularly, a subject of the present invention is an isolated orpurified polynucleotide encoding a polypeptide involved, in itswild-type form, in synaptogenesis. The polynucleotide of the presentinvention is characterized in that at least one mutation in the nucleicacid sequence of said polynucleotide is associated with the developmentof neurological diseases and/or with a predisposition to the developmentof mental disorders or psychiatric diseases.

Preferably, the present invention relates to a polynucleotide encoding aprotein belonging to the family of human neuroligins (HNLs), and moreparticularly the HNL4X protein (previously called HNL4) and itsfunctional homolog HNL4Y (previously called HNL5) encoded by a gene onthe Y chromosome.

The present invention also relates to a polynucleotide encoding themouse protein MNL4, which is the orthologs of the HNL4X and HNL4Yproteins.

According to another subject, the present invention is directed towardan isolated or purified polypeptide, characterized in that it is encodedby a polynucleotide as defined above. More particularly, the polypeptideaccording to the present invention is characterized in that it isinvolved in synaptogenesis, and in that the presence of at least onemutation in the amino acid sequence of said polypeptide is associatedwith a predisposition to the development of mental disorders orpsychiatric diseases.

According to another subject, the present invention proposes a methodfor detecting biochemical disorders which alter synapse formation,and/or a predisposition to the development of psychiatric pathologiesand/or a mental disease, comprising at least one of the following steps:

-   detecting a mutation in the sequence of a polynucleotide as defined    above, in the sequence of a fragment of said polynucleotide or in    the sequence of a messenger RNA of said polynucleotide;-   detecting the presence of a polypeptide as defined above;-   detecting a mutation in a polypeptide as defined above;-   measuring the activity of a polypeptide as defined above or the    interaction thereof with one of its protein partners.

Another object of the invention is to provide a kit for detectingbiochemical disorders which alter synapse formation, and/or apredisposition to the development of psychiatric pathologies and/or amental disease, and/or for diagnosing a mental disease, the kitcomprising at least one of the elements chosen from the group consistingof: a probe, an antibody, a reagent and a solid support for:

-   -   i) detecting a mutation in the sequence of a polynucleotide as        defined above, in the sequence of a fragment of said        polynucleotide or in the sequence of a messenger RNA of said        polynucleotide; and/or    -   ii) measuring the biological activity of a polypeptide as        defined above or the interaction thereof with one of its protein        partners.

The invention also relates to the use of a nonmutated polynucleotideencoding a protein involved, in its wild-type form, in synaptogenesis,for treating or preventing biochemical pathologies or mental diseases.

The invention also relates to the use of a nonmutated polypeptideinvolved, in its wild-type form, in synaptogenesis, for treating orpreventing biochemical pathologies or mental diseases.

The present invention also proposes a method for sorting molecules whichmakes it possible to modulate the biological activity of the polypeptideencoded by the polynucleotide defined above or the biological activityof the polypeptide defined above, comprising:

-   -   a) bringing said polypeptide or a recombinant cell containing it        into contact with a molecule capable of modulating its        biological activity;    -   b) measuring the biological activity of said polypeptide or its        interaction with one of its protein partners; and    -   c) evaluating the activity measured in step b) relative to a        measurement of the biological activity of said polypeptide in        the absence of said molecule.

Another subject of the present invention is directed. toward a methodfor treating a mental or neurological disease, comprising the insertioninto at least one portion of the cells from an affected patient of apolynucleotide encoding a polypeptide as defined above.

The present invention also proposes a method for transforming stem cellsfrom a patient exhibiting a mutation of a gene encoding a proteininvolved in synaptogenesis, characterized by

-   -   a) the use of stem cells from said patient;    -   b) the insertion into the genome of said stem cells of a        polynucleotide as defined above; and    -   c) the reimplantation into the patient of cells transformed        according to step b).

The invention also relates to a cloning or expression vector comprisingone of the polynucleotides of the invention or a fragment thereof; ahost cell containing a polynucleotide and/or a vector according to theinvention; or purified monoclonal or polyclonal antibodies whichrecognize specifically at least one of the polynucleotides of theinvention and/or at least one of the polypeptides of the invention.

The invention is also directed toward a composition containing at leastone element chosen from the group consisting of a) a polypeptide, b) apolynucleotide, c) a vector, d) a host cell and e) an antibody, and apharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the region of chromosome Xp22.3 containing the HNL4X gene.

FIG. 2 shows a protein alignment for human neuroligins (HNL1-SEQ IDNO:64; HNL2-SEQ ID NO:65; HNL3-SEQ ID NO:66; HNL4-SEQ ID NO:67; HNL5-SEQID NO:6).

FIG. 3 is a diagram which shows the protein architecture of the synapsewith the location and the known partners of neuroligins.

FIGS. 4A and 4B represent a diagram which shows the chromosomal locationand the evolution of the HNL4X and HNL4Y genes.

FIG. 5 is a diagram which shows the genomic structure of the humanneuroligin genes.

FIGS. 6A and 6B show the result of SSCP (single strand conformationalpolymorphism) analysis of a mutation of the gene encoding the HNL4Xprotein and also the sequence of this mutation.

FIG. 7 is a diagram which shows the location of the HNL4X protein andthe effects of the mutation on HNL4X.

FIG. 8A-1 to FIG. 8A-66 represent the genomic sequence (SEQ ID NO:1) ofthe wild-type (nonmutated) human HNL4X gene.

FIG. 8B-1 to FIG. 8B-2 represent the nucleic acid sequence (SEQ IDNO:16) of exon 1 of FIG. 8A.

FIG. 9A-1 to FIG. 9A-2 represent the complementary DNA sequence (SEQ IDNO:2) of the wild-type (nonmutated) human HNL4X gene.

FIG. 10 represents the amino acid sequence (SEQ ID NO:3) of thewild-type (nonmutated) human HNL4X protein.

FIG. 11A represents the genomic sequence (SEQ ID NO:4) of the humanHNL4Y gene.

FIG. 11B represents the nucleic acid sequence (SEQ ID NO:17) of exon 1of FIG. 11A.

FIG. 12 represents the complementary DNA (cDNA) sequence (SEQ ID NO:5)of the wild-type human HNL4Y gene.

FIG. 13 represents the amino acid sequence (SEQ ID NO:6) of thewild-type human HNL4Y protein.

FIG. 14 represents the complementary DNA (cDNA) sequence (SEQ ID NO:7)of an alternative transcript of the wild-type human HNL4Y gene.

FIG. 15 represents the amino acid sequence (SEQ ID NO:8) correspondingto the alternative sequence of FIG. 14.

FIG. 16 represents the amino acid sequence (SEQ ID NO:9) of the mutatedhuman HNL4X protein.

FIG. 17 shows the chromosomal location of the HNL3, HNL4X and HNL4Ygenes and the pedigree of a family exhibiting a mutation in HNL4X in anautistic boy and a boy suffering from Asperger syndrome.

FIG. 18 is a diagram which shows the conservation of the HNL3 mutations(Neuroligins-SEQ ID NOS:68-74; Acetylcholine esterase-SEQ ID NO:75-88;Butyrylcholine esterase-SEQ ID NOS:89-92; HNL3 N796S-SEQ ID NOS93-98).

FIG. 19A shows the nucleic acid sequence of the cDNA of MNL4 (SEQ IDNO:62) (orthologs of HNL4X and HNL4Y).

FIG. 19B represents the amino acid sequence of the MNL4 protein (SEQ IDNO:63).

FIG. 20 is a diagram which shows the genomic structure of the HNL1, HNL2and HNL3 genes, and also the location of the mutations observed in HNL3.

FIG. 21 is a diagram which shows the genomic structure of HNL4X andHNL4Y.

FIG. 22 shows a portion of the amino acid sequence (SEQ ID NO:10) of theHNL3 protein mutated at position 451.

FIG. 23 shows another portion of the amino acid sequence (SEQ ID NO:11)of the HNL3 protein mutated at position 796.

FIG. 24 represents the complementary DNA (cDNA) sequence (SEQ ID NO:12)of the wild-type HNL3 transcript.

FIG. 25 represents the complementary DNA (cDNA) sequence (SEQ ID NO:13)of the mutated HNL3 transcript.

FIG. 26 represents the amino acid sequence (SEQ ID NO:14) of thewild-type human HNL3 protein.

FIG. 27 represents the amino acid sequence (SEQ ID NO:15) of the mutatedhuman HNL3 protein.

DETAILED DESCRIPTION OF THE INVENTION

The originality of the present invention relates to the identificationof the genomic sequence of the HNL4X gene located at Xp22.3 and of afunctional homolog HNL4Y placed on the Y chromosome, located at Yq11.22,and also their murine orthologs MNL4.

The invention also relates to the identification of the involvement ofthe proteins of synaptogenesis, in particular HNL3 and HNL4, in thedevelopment of mental disorders or psychiatric diseases such as autism.

1. Polypeptide and Polynucleotide

According to a first aspect, the present invention is directed toward anisolated or purified polypeptide which, in its wild-type (i.e.nonmutated) form, is involved in synaptogenesis, in which at least onemutation in the amino acid sequence is associated with the developmentof neurological diseases and/or with a predisposition to the developmentof mental disorders or psychiatric diseases.

The expression “mental disorders or psychiatric diseases” is intended tomean diseases such as autism, Asperger syndrome, schizophrenia and ADHD(attention deficit hyperactivity disorder) syndrome.

Preferably, the polypeptide consists of a cell adhesion protein, morepreferably of a protein belonging to the human neuroligin family, andeven more preferably the polyeptide consists of the HNL3 protein, HNL4Xor the HNL4Y protein. Advantageously, when the polypeptide is HNL3, itcomprises an amino acid sequence according to SEQ ID NO:14 and sequencesof at least 20, of at least 50 and of at least 100 consecutive aminoacids or more derived from SEQ ID NO:14. When the polypeptide of theinvention is HNL4X or the HNL4Y protein, it comprises a sequence chosenfrom the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8 andthe sequences of at least 20, of at least 50 and of at least 100consecutive amino acids or more derived from SEQ ID NO:3, SEQ ID NO:6 orSEQ ID NO:8.

The invention also relates to the “mutated” polypeptides and thepolypeptides “derived” from the wild-type protein, preferably aneuroligin such as HNL3, HNL4X or HNL4Y.

The term “polypeptide derived” from a wild-type protein or “variant” ofa wild-type protein is intended to mean all peptides which have apeptide sequence substantially identical, at least in part, to thepeptide sequence of the wild-type protein. They may, for example, bechemically modified polypeptides having a peptide sequence 100%identical to a portion of the wild-type protein. They may also be hybridpolypeptides having a first portion 100% identical to a first portion ofthe wild-type protein and a second portion in no way/partially identicalto a second portion of the wild-type protein. They may also bepolypeptides having complete/partial homology with a portion of thewild-type protein.

The term “mutated” polypeptides derived from a wild-type protein isintended to mean all peptides which have been obtained followingmodification of said wild-type protein, whether this is modification byaddition, deletion or substitution of one or more of the amino acids ofthe wild-type protein. It may also be a modification introduced by theaddition of carbon chains attached to at least one of the amino acids ofthe wild-type protein or to at least one of the amino acids of thepeptides for which there exists a substitution or a modification of oneof the amino acids compared to the wild-type protein. More particularly,the present invention covers the peptides which derive from the humanprotein HNL3, HNL4X or HNL4Y.

According to a preferred embodiment, and when the polypeptide is amutated HNL3 according to the present invention, it has SEQ ID NO:10,SEQ ID NO:11or SEQ ID NO:15, and a sequence of at least 20, of at least50 and of at least 100 consecutive amino acids or more derived from SEQID NO:10, SEQ ID NO:11 or SEQ ID NO:15. When the polypeptide is amutated HNL4X or a mutated HNL4Y according to the present invention, ithas SEQ ID NO:9 or a sequence of at least 20, of at least 50 and of atleast 100 consecutive amino acids or more derived from SEQ ID. NO:9.

The invention is also directed toward the polypeptides (and thefragments thereof) which are encoded by the nucleotide sequencesmentioned hereinafter.

In the context of the present invention, the term “polypeptide” isdefined as being any peptide or protein comprising at least two aminoacids linked by a modified or unmodified peptide bond. The term“polypeptide” refers to short-chain molecules such as peptides,oligopeptides or oligomers or to long-chain molecules such as proteins.A polypeptide according to the present invention can comprise modifiedamino acids. Thus, the polypeptide of the present invention can also bemodified by a natural process such as post-transcriptional modificationsor by a chemical process. Some examples of these modifications are:acetylation, acylation, ADP-ribosylation, amidation, covalent bondingwith flavine, covalent bonding with a heme, covalent bonding with anucleotide or a nucleotide derivative, bonding with a lipid or a lipidderivative, covalent bonding with a phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, cysteine moleculeformation, pyroglutamate formation, formylation, gamma-carboxylation,hydroxylation, iodination, methylation, oxidation, phosphorylation,racemization, hydroxylation, etc. Thus, any modification of thepolypeptide which does not have the effect of eliminating thebiochemical characteristics of the polypeptide of origin, i.e. theability to form functional synapses, is covered within the scope of thepresent invention.

According to a related aspect, the invention is directed toward anisolated or purified polynucleotide encoding a polypeptide as definedabove, and more particularly an isolated or purified polynucleotideencoding a polypeptide involved in synaptogenesis, in which at least onemutation of this polypeptide is associated with the development ofneurological diseases and/or with a predisposition to the development ofmental diseases or psychiatric diseases.

The term “isolated or purified” is intended to mean the molecules whichhave been altered, by man, from their natural state, i.e., if such amolecule exists naturally, it has been changed and/or removed from itsinitial environment. For example, a polynucleotide or a polypeptidenaturally present in a living organism is not “isolated”. However, thesame polynucleotide or polypeptide, when separated from its normalenvironment and/or obtained by cloning, amplification and/or by chemicalsynthesis, is considered according to the present invention as being“isolated”. Moreover, a polynucleotide or polynucleotide which isintroduced into an organism by transformation or genetic manipulation orby any other method of recombination is “isolated” even if it is presentin said organism.

The term “polynucleotide” is intended to mean any DNA or RNA molecule orsequence having two nucleotides or more, including the nucleic acidsequences encoding an entire gene. The term “polynucleotide” encompassesall nucleic acid molecules which are in the natural or artificial state.This includes DNA molecules, RNA molecules, cDNAs, expressed sequences(ESTs), artificial sequences and all the fragments thereof. It goeswithout saying that the “derived”, “variant” and “mutated” definitionsalso apply to the polynucleotides according to the present invention.Any polynucleotide which has been chemically, enzymatically ormetabolically modified but which has conserved the biochemicalproperties of the polypeptide of origin, i.e. which has conserved itsability to form functional synapses, is included in the scope of thepresent invention.

According to a preferred embodiment, when the polynucleotide accordingto the invention encodes an HNL3 protein or a fragment of this protein,the latter advantageously comprisses SEQ ID NO: 14. Preferably, thepolynucleotide comprises a sequence chosen from the group consisting of: SEQ ID NO: 12 and the sequences of at least 20, of at least 50 and ofat least 100 consecutive nucleotides or more derived from SEQ ID NO: 12.

According to a preferred embodiment, when the polynucleotide accordingto the invention encodes an HNL4X or HNL4Y protein or a fragment of thisprotein, the latter advantageously comprises SEQ ID NO:3, SEQ ID NO:6 orSEQ ID NO:8. Preferably, the polynucleotide comprises a sequence chosenfrom the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17 and the sequences of atleast 20, of at least 50 and of at least 100 consecutive nucleotides ormore derived from these sequences.

According to another embodiment, the polynucleotide encodes anonfunctional mutated protein. Preferably, the polynucleotide encodes amutated HNL3 or mutated HNL4X protein. When the protein is HNL4X, thepolynucleotide is mutated such that the mutation causes earlytermination of the protein. More preferably, the polynucleotide of theinvention comprises SEQ ID NO:1 and the mutation is an insertion of athymine at position 1186, from position 465 of FIG. 9 (ORF). Thismutation causes the production of a defective protein lacking itstransmembrane portion since this mutation causes early termination ofthe protein (D396stop). When the mutated protein is HNL3, the mutationcauses a modification of the protein sequence such as an amino acidsubstitution at position 451 and/or 796 of FIG. 18 or 21. Moreparticularly, the mutation produced at position 451 consists of thesubstitution of an arginine with a cysteine, while the mutation producedat position 796 consists of the substitution of an asparagine with aserine. This amino acid, arginine R451, is located in the acetylcholineesterase domain of the neuroligins and is extremely conserved in all theneuroligins sequenced to date and in fish, bird and reptileacetylcholine esterases (FIGS. 6A and 6B).

The polypeptides and polynucleotides according to the present inventioncan be prepared by any suitable method. They can in particular beobtained by chemical synthesis, but it is also possible to obtain thembiologically, using in particular various vectors in suitable cellcultures, as will be described hereinafter. The peptides according tothe present invention can be in deglycosylated or glycosylated form, ifthis is necessary. Those with knowledge in the field of the inventionwill be able to obtain various polynucleotides/polypeptides and willalso be able to determine which, among the polynucleotides/polypeptidesobtained, are those which have an appropriate biological activity.

2. Vector, Antibody and Cell

According to another aspect, the invention relates to any vector(cloning and/or expression vector) and any cellular host (procaryotic oreukaryotic) transformed with such a vector, and comprising theregulatory elements for expression of the nucleotide sequence encoding apeptide according to the invention.

According to another aspect, a subject of the invention is a method forpreparing a peptide of the invention, by transformation of a cellularhost using an expression vector (plasmid, cosmid, virus, etc.)comprising the DNA sequences encoding the peptides of the invention,followed by culturing of the cellular host thus transformed, andrecovery of the peptide from the culture medium. The use of vectors forthe expression of proteins and peptides in the cells of host, inparticular humans, is well known and will not be described in furtherdetail.

The polypeptides and polynucleotides of the present invention can alsobe used to prepare polyclonal or monoclonal antibodies capable ofbinding (preferably specifically) to at least onepolypeptide/polynucleotide which is a subject of the invention. Thepresent invention is therefore also directed toward such purifiedantibodies which can be obtained by very well-known techniques such as,for example, the technique described by Kohler and Milstein (Continuouscultures of fused cells secreting antibody of predefined specificity,Nature, 1975, 262:495-497). According to a preferred embodiment of theinvention, the antibodies are of the “humanized” type. Those skilled inthe field, by virtue of their general knowledge, will know how toprepare these types of antibodies.

In the context of the present invention, the term “vector” refers to apolynucleotide construct designed to be transfected into various celltypes. As a result, these vectors are directed toward expression vectorsdesigned for the expression of a nucleotide sequence in a host cell;cloning vectors designed for the isolation, propagation and replicationof inserted nucleotides; viral vectors designed for the production ofrecombinant virus or of viral particle (viral-like particle); or shuttlevectors which comprise attributes of more than one vector.

3. Methods and Process for Use

According to another aspect, the invention relates to the treatment orprevention of biochemical pathologies or mental diseases such as autismor Asperger syndrome. More particularly, the invention is directedtoward the use of a nonmutated polynucleotide encoding a proteininvolved in synaptogenesis. Preferably, the protein consists of a celladhesion protein, more preferably of a protein belonging to the humanneuroligin family, and even more preferably the polypeptide consists ofthe HNL3 protein, HNL4X or the HNL4Y protein. Examples of nonmutatedpolynucleotides are given above.

The invention is also directed toward a method of treatment comprisingthe insertion into at least one portion of the cells from an affectedpatient of a polynucleotide encoding a polypeptide involved insynaptogenesis, such as the HNL3 or HNL4X protein. Preferably, the cellsinto which the polynucleotide is inserted are stem cells. Examples ofsatisfactory polynucleotides are given above.

According to a related aspect, the invention is directed toward a methodfor transforming stem cells from a patient exhibiting a mutation of agene encoding a protein involved in synaptogenesis, the methodcomprising:

-   -   a) the use of stem cells from the patient;    -   b) the insertion into the genome of the stem cells, of a        polynucleotide encoding a functional polypeptide involved in        synaptogenesis, such as the HNL3 or HNL4X protein; and    -   c) the reimplantation into the patient of cells transformed        according to step b).

Those skilled in the field will be able to adapt the treatment methodsmentioned above and to determine, according to several factors, thepolynucleotides which should be used, the means for inserting them intothe cells and the method and the amount of polynucleotides or of cellswhich should be administered. Among the factors which can influencetheir choices are: the nature of the treatment; the exact sequence ofthe polynucleotides; the stage of the disease; the condition, the ageand the weight of the patient, etc.

According to another aspect, the invention also relates to a method fordetecting biochemical disorders which alter synapse formation,stabilization and/or recognition, a predisposition to the development ofpsychiatric pathologies and/or a mental disease such as autism orAsperger syndrome.

Thus, the method comprises at least one of the following steps:

-   -   detecting a mutation in the sequence of a gene encoding a        protein involved in synaptogenesis, in the sequence of a        fragment of this gene or in the sequence of a messenger RNA of        this gene;    -   detecting the presence of a protein involved in synaptogenesis;    -   detecting a mutation in a protein involved in synaptogenesis;    -   measuring the biological activity of a protein involved in        synaptogenesis or its interaction with one of its protein        partners. A method for measuring such an interaction is, for        example, described in Ichtchenko et al. (J. Biol. Chem., 1996,        271(5):2676-82) or Grifman et al. (Proc.

Natl. Acad. Sci. USA, 1998, 95(23):13935-40).

According to a preferred embodiment, the method comprises:

-   -   a) amplifying a gene encoding a protein involved in        synaptogenesis, amplifying a fragment of said gene or amplifying        a messenger RNA of said gene; and    -   b) detecting a mutation in the sequence of said gene, in the        sequence of said fragment or in the sequence of said messenger        RNA.

A related aspect of the method of the invention concerns a kit (set) fordetecting biochemical disorders which alter synapse formation, apredisposition to the development of psychiatric pathologies and/or amental disease, and/or for diagnosing a mental disease. According to apreferred embodiment, the kit comprises at least one of the elementschosen from the group consisting of: a probe, an antibody, a reagent anda solid support, these elements allowing:

-   i) detection of a mutation in the sequence of a gene encoding a    protein involved in synaptogenesis, in the sequence of a fragment of    this gene or in the sequence of a messenger RNA of this gene; and/or-   ii) measurement of the biological activity of a protein involved in    synaptogenesis or of its interaction with one of its protein    partners.

Preferably, the gene to which reference is made in the method and thekit encodes, in its wild-type form, a cell adhesion protein, morepreferably a protein belonging to the human neuroligin family, and evenmore preferably the HNL3 protein, the HNL4X protein or the HNL4Yprotein.

Advantageously, the gene encodes, in its wild-type form, a proteincomprising SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:14. Morepreferably, the gene comprises SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:12.

Knowledge of the gene involved in a predisposition to the development ofautism or of Asperger syndrome opens the door to the discovery of novelmolecules for preventing, controlling or treating the disease. Thus,according to another aspect, the invention is directed toward a methodfor sorting molecules which can make it possible to modulate thebiological activity of a polypeptide encoded by the polynucleotide asdefined above, or the biological activity of the polypeptide as definedabove. According to a preferred embodiment, the sorting methodcomprises:

-   -   a) bringing said polypeptide into contact with a molecule        capable of modulating its biological activity;    -   b) measuring the biological activity of said polypeptide; and    -   c) evaluating the activity measured in step b) relative to a        measurement of the biological activity of said polypeptide in        the absence of said molecule.

4. Compositions

The present invention also relates to the use of these polypeptides andof the polynucleotides encoding them, for preparing therapeuticcompositions that are useful in the treatment of a mental orneurological disease, such as autism, Asperger syndrome, schizophreniaor ADHD syndrome.

In a preferred embodiment, the composition of the present invention alsocontains a pharmaceutically acceptable vehicle, and an element chosenfrom the group consisting of:

-   -   a polynucleotide according to the present invention;    -   a polypeptide according to the present invention;    -   an antibody according to the present invention;    -   a vector according to the present invention; and    -   a host cell according to the present invention.

The compositions according to the present invention can be in any solidor liquid form that is usual for pharmaceutical administration, i.e.,for example, liquid administration forms or administration formsconsisting of a gel, or any other support allowing, for example,controlled release. Among the compositions which can be used, mentionmay in particular be made of injectable compositions more particularlyintended for injections into the blood circulation in humans.

Those skilled in the field will be able to prepare pharmaceuticallyacceptable compositions and to determine, according to several factors,the preferred mode of administration and the amount which should beadministered. Among the factors which can influence their choices are:the nature of the treatment; the exact nature of the active ornon-active ingredients which make up the composition; the stage of thedisease; the condition, the age and the weight of the patient, etc.

The examples hereinafter will make it possible to demonstrate othercharacteristics and advantages of the present invention.

EXAMPLES

The examples which follow serve to illustrate the extent of the use ofthe present invention and not to limit its scope. Modifications andvariations can be made thereto without departing from the spirit or fromthe scope of the invention. Although other methods or productsequivalent to those which are found above may be used for testing orimplementing the present invention, the preferred materials and methodsare described.

Introduction

A locus for predisposition to autism has been suggested in Xp22.3 by theobservation of several independent and de novo chromosomal deletions inthis region. The size of the critical region deleted in these patientswas approximately 10 Mb, delimited by DXYS232X (6 cM) and DXS7103 (16cM). In support of these results, the overall analysis of the genomecarried out by the Paris study (Philippe et al., 1999) indicates thatthe maximum LOD for the X chromosome is located in this region (11 cM).

Within this interval, we have identified the human neuroligin 4 (HNL4X)gene encoding a new member of the neuroligin family (Scheiffele et al.,2000; Song et al., 1999). These cell adhesion molecules possess ahomology with acetylcholine esterases and are specifically located inthe postsynaptic membrane of excitatory synapses (Song et al., 1999).They are essential factors for the formation of functional synapsessince the expression of neuroligins in HeLa cells or kidney cells cantrigger the development of presynaptic structures in neurons which arein contact (Scheiffele et al., 2000). We have identified five HNL genesin the human genome, located on chromosomes 3q26 (HNL1), 17p13 (HNL2),Xq13 (HNL3), Xp22.3 (HNL4X) and Yq11.2 (HNL4Y). Neuroligin phylogenysuggests that HNL3 is the common ancestor of HNL4X and HNL4Y. Theexpression profile for the HNLs has been determined by specific RT-PCRsin various adult (male and female) brain tissues. Expression of theHNL1-3 genes and of their alternative transcripts is found in all brainregions. HNL4X and HNL4Y are expressed at similar levels in the malebrain without significant differences between the various tissues. Asexpected, HNL4Y is not expressed in the female brain, whereas HNL4X isexpressed.

Example 1 Characterization of the HNL4X and HNL4Y Genes and theirInvolvement in Psychiatric Syndromes

The phylogenetic study shows that the HATL4X genes located on the Xchromosome and the HNL4Y gene located on the Y chromosome began todiverge approximately 40 million years ago, during the evolution ofprimates. While all the genes of the Y chromosome which diverged at thisdate became pseudogenes (inactive genes), HNL4Y is strongly conserved(table 1).

TABLE 1 Conservation of the HNL4X and HNL4Y genes during evolution DNAProtein Compared diver- diver- sequence gence gence (nucleo- Gene pairKs KA Ks/KA (%) (%) tides) Group 4 GYG2/GYG2P* 0.11 0.06 1.8 7 12 525ARSD/ARSDP* 0.09 0.07 1.3 7 13 846 ARSE/ARSEP* 0.05 0.04 1.2 4 9 615PRKX/Y 0.07 0.03 2.3 5 8 1020 HNL4X/4Y 0.079 0.012 6.456 3 2 2451STS/STSP* 0.12 0.10 1.2 11 18 852 KAL1/KALP* 0.07 0.06 1.2 6 12 1302AMELX/Y 0.07 0.07 1.0 7 12 576 Group 3 TB4X/Y 0.29 0.04 7.3 7 7 135EIF1AX/Y 0.32 0.01 32 9 2 432 ZFX/Y 0.23 0.04 5.8 7 7 2394 DFFRX/Y 0.330.05 6.6 11 9 7671 DBX/Y 0.36 0.04 9.0 12 9 1932 CASK/CASKP* 0.24 0.221.1 15 32 156 UTX/Y 0.26 0.08 3.3 12 15 4068 Group 2 UBE1X/Y 0.58 0.078.3 16 13 693 SMCX/Y 0.52 0.08 6.5 17 15 4623 Group 1 RPS4X/Y 0.97 0.0519 18 18 792 RBMX/Y 0.94 0.25 3.8 29 38 1188 SOX3/SRY 1.25 0.19 6.6 2829 264 PCDHX/Y 0.006 0.008 0.809 1 2 2850

This table includes the synonymous substitution rates (KS) andnonsynonymous substitution rates (KA) of all the known genes of the Xchromosome having homology on the Y chromosome. The KS/KA ratio is anindication of the gene conservation. KS is the rate of synonymoussubstitution per synonymous site which represents the modificationswhich do not change the sequence of the protein. KA is the rate ofnonsynonymous substitution per nonsynonymous site which represents themodifications which change the sequence of the protein. If the KS/KAratio=1, then the gene is not conserved since there are as manysynonymous modifications as there are nonsynonmyous modifications. Thisis the case for the X/Y pairs having a nonfunctional pseudogene on the Ychromosome (for example, KALl/KALP*). If KS/KA>1, then the gene variesin the course of evolution but the protein is well conserved. This isthe case for the HNL4/5 pair indicating that the genetic variationbetween these genes is subjected to a selection pressure which conservesthe protein sequences of HNL4X and HNL4Y.

For the detection of mutations on the HNL4X gene, the following stepswere used:

Materials and Methods

Identification of the Sequence of the HNL4X, HNL4Y and MNL4 Genes

The HNL4X and HNL4Y genes were isolated by computer analysis of thesequences of the Xp22.3 and Yq11.22 region and amplification of thecomplete transcripts from brain mRNA.

Computer Analysis

A systematic study of the genes of the Xp22.3 region, close to theDXS996 microsatellite, was carried out using the human genome sequencingdata available through publicly available databases. The DXS996microsatellite is the genetic marker which shows the most significantlinkage with autism in the analysis by Philippe et al. (1999, mentionedabove). We identified that this genetic marker was located in a putativegene (KIAA1260, and that a putative homolog, KIAA0951, of this geneexisted, located on the Y chromosome. The partial sequence of the cDNAsof the genes encoding KIAA1260 and KIAA0951 was deduced from the genomicsequence. A (BLAST) sequence alignment analysis and a phylogenetic treegrouping together the other human neuroligins were effected so as todefine that KIAA1260 and KIAA0951 are new members of the neuroliginfamily which we henceforth call HNL4X and HNL4Y.

Analysis of the HNL4X and HNL4Y Transcripts

Total RNA from human brains coming from various men (n=5) and women(n=5) was isolated from biopsies of frontal cortex. The complete cDNAsof the HNL4X and HNL4Y mRNAs were reverse transcribed, amplified anddirectly sequenced. The oligonucleotides used for the amplification andsequencing are indicated in tables 2 and 3.

Sequencing of the HNL4X and HNL4Y Genes in Autistic and AspergerIndividuals

Each exon of the HNL4X and HNL4Y genes was amplified and sequenced fromthe genomic DNA. The name and the sequence of each primer are indicatedin table 3.

TABLE 2 Names and sequences of the primers used to amplify and sequencethe HNL4X and HNL4Y cDNAs Exons HNLXY1 ACCCCGCGTGAAGATGAAATG SEQ IDNO:18 1-6 HNLXYE6dR GAGGGATAGGARGGGAAATAG SEQ ID NO:19 Exons HNLXYE2FGGATGTGGATGCAGATTTGAA SEQ ID NO:20 2-5 HNLXY4 GCTCTGAATGATGGCCTTCTG SEQID NO:21 Exons HNLXY10 TCCTGGATCAGATTCAAGCAC SEQ ID NO:22 4-6 HNLXYE6dRGAGGGATAGGARGGGAAATAG SEQ ID NO:23 Exons HNLXYE2F GGATGTGGATGCAGATTTGAASEQ ID NQ:24 2-6 HNLXYE6dR GAGGGATAGGARGGGAAATAG SEQ ID NO:25

For the degenerate primers, use of the universal code: M(AC), R(AG),W(AT), S(CG), Y(CT), K(GT), V(ACG), H(ACT), D(AGT), B(CGT), N(ACGT)

TABLE 3 Names and sequences of the primers used to sequence the HNL4Xand HNL4Y genes SEQ ID Exons Primers Sequences NO: Exon 1a HNLXYE1aFGAAACAACGAATTTCCTCCAAA 26 HNL4X HNLXYE1aR AGTGAGGCTTTCCATCCTTTGC 27 Exon1b HNLXE1F ATTCTTTAAGAAAACTGTCAGC 28 HNL4X HNLXYE1RCACGGGAAAGGGGTGCATGGA 29 Exon 1 HNLYE1F GGGGTGCTTCTTTTGGGAGGCT 30 HNL4YHNLXYE1R CACGGGAAAGGGGTGCATGGA 31 Exon 2 HNLXYE2F GGATGTGGATGCAGATTTGAA32 HNLX/Y HNLXE2Rbis GTATTGTTTTCTGTTCCAGTG 33 Exon 3 HNLXYE3FTGTGTTTCCGTACTTGGCTTT 34 HNL4X/Y HNLXYE3R GCTTAGTCATTCACATGATGAA 35 Exon4 HNLXYE4F ACCAAAAATCTCTTGTGTTCT 36 HNL4x HNLXYE4R TTCTTGGTTCAGGGTATTTGC37 Exon 4 HNLYE4F AACAAAAATGTCCTGTGTTCT 38 HNL4Y HNLXYE4RTTCTTGGTTCAGGGTATTTGC 39 Exon 5 RNLXYE5dF TGTCCRCAATTTTGCACCTGC 40HNL4X/Y HNLXYE5dR AGGAYAGTGATACCCCAACA 41 Exon 6 HNLXYE6FbisAGAGCAGATTGTAACTTCCTG 42 RNL4X/Y HNLXYE6dR GAGGGATAGGARGGGAAATAG 43

For the degenerate primers, use of the universal code: M(AC), R(AG),W(AT), S(CG), Y(CT), K(GT), V(ACG), H(ACT), D(AGT), B(CGT), N(ACGT).

TABLE 4 Names and sequences of the primers used for the amplification ofthe MNL4 cDNA (57BL6 mouse) Primer Sequence SEQ ID NO: MNL4F8CGTGACGAAACAGGAAGTGACC 44 MNL4R8 GTAGCCAAGGCCCCTGCATGTC 45

TABLE 5 Names and sequences of the primers used for the amplification ofMNL4 in three PCRs of approximately 1 kb Primer Sequence SEQ ID NO:MNL4F8 CGTGACGAAACAGGAAGTGACC 46 MNL4R2 AGCCGAAGACGGTGACGCGGTC 47MNL4F12 AGGAAGCCGGTCATGGTTTACA 48 MNL4R5 ACGCTCAGCTCCGTCGAGTAGT 49MNL4F14 AGACGCTCGTGGCGCTCTTCAC 50 MNL4R8 GTAGCCAAGGCCCCTGCATGTC 51

TABLE 6 Names and sequences of the primers used for the amplification ofHNL3 PRIMER SEQUENCE SEQ ID NO: RNL3E2F CCTATTGGGCTGATGCTGTGACC 52HNL3E2R AGGGCACACAACCACATGCAAG 53 HNL3E3Fbis TTGAGCTCCAGGTTGAGCAACC 54HNL3E3RBIS CCCCTTGCGAAGCCAGTCTTCC 55 HNL3E4F CTGCGTGCTCATTCTCTATTCC 56HNL3E4R GTAGAAGAGAGCTGGCCGATTC 57 HNL3E5F ATGGCTATGTGTGACACGACAG 58HNL3E5R GGAAGATGAGTGAAGGGGTACC 59 HNL3EGF TTTCCTCATCCAGATAGAGTGG 60HNL3E6R CATGTGTTCCTGGATCTGGGAG 61

In order to test this gene in autistic individuals, the genomicstructure of the various HNLs was defined and the coding portions (Exon2-Exon 6) were amplified.

Thus, 140 autistic boys and 18 autistic girls were tested for most ofthe HNL4X/4Y coding portion.

In a Swedish family with two affected brothers, one with autism and theother exhibiting Asperger syndrome, an additional thymidine wasidentified at nucleotide 1186 of the HNL4X gene, creating a stop codon(FIG. 17). This mutation (D396stop) is located in the esterase domain,producing a premature termination of the protein and deleting thetransmembrane domain. This change is inherited from the mother, but isabsent in the maternal grandmother, in the two maternal aunts and in theunaffected child, indicating the de novo status of this mutation in themother of the affected boys. In addition, this mutation was not found in350 controls (250 women and 100 men).

In addition, the boy/girl ratio, which is four for autism and nine forAsperger syndrome, corroborates this observation according to whichHNL4X/4Y influences synaptogenesis and the mutation of HNL4X/4Yconstitutes a factor for predisposition to mental diseases, inparticular autism and Asperger syndrome.

The identification of this stop mutation in a primate-specific genecarried by the X chromosome in two autistic individuals, and involved insynaptogenesis, is one of the first functional mutations identified in apsychiatric disease. This mutation is also the first mutation describedwhich is associated with autism without any other clinical sign (fragileX, tubercular sclerosis, etc.).

Example 2 Characterization of the HNL3 Gene and of Its Involvement inPsychiatric Syndromes

During the search for mutations in HNL3, the ancestral gene for HNL4X/Ylocated in the Xq13 region, in two independent families, two amino acidchanges located in highly conserved regions of the protein wereidentified. One of the two families is very similar to the first familymutated in HNL4X. The two affected brothers, the first affected withautism and the second with Asperger syndrome, received the mutation fromtheir mother. Interestingly, the mother also has a brother with Aspergersyndrome and other relatives with psychiatric disorders. The mutation(R451C) is located in the esterase domain of the protein and concerns anamino acid that is conserved during evolution since it is present in allneuroligins (including in D. melanogaster) and in all the mammalian,fish, reptile and bird acetylcholine esterases sequenced to date (seeFIG. 6). The mutation is absent in 200 controls (100 women and 100 men).These results support the role of neuroligins in the etiology of mentaldisorders or psychiatric diseases such as autism and Asperger syndrome.MNL4, the orthologs of HNL4X in mice, was also identified. This new geneshould make it possible to understand the deficiency induced by amutation such as the HNL4X mutation in autism (see FIG. 19).

The final examination of the genome carried out on Finnish families withmembers suffering from autism (Auranen, et al., 2002) identified twovery significant linkage peaks at 3q26 (exactly where HNL1 is located)and at Xq13-21 (exactly where HNL3 is located). The Xp22.3 region,containing HNL4X, is also deleted in two patients with schizophrenia. Itis therefore possible that neuroligins are also responsible forsusceptibility to this syndrome (schizophrenia affects 1% of thepopulation).

1. A method of diagnosing autism linked to a mutation in thepolynucleotide of SEQ ID NO: 12 or the polypeptide of SEQ ID NO: 14, ora propensity therefor, in a human, wherein said mutation results inaltered synapse formation, wherein said method comprises (a) detecting amutation in (i) the polynucleotide of SEQ ID NO: 12, wherein saidmutation results in a mutation in the polypeptide encoded thereby atposition 451 or 796, or a combination thereof, or (ii) the polypeptideof SEQ ID NO: 14, wherein said mutation is at position 451 or 796, or acombination thereof, wherein said detecting comprises comparing thesequence of the polynucleotide or polypeptide obtained from said humanwith the polynucleotide of SEQ ID NO: 12 or the polypeptide of SEQ IDNO: 14 and identifying mutations in said polynucleotide or polypeptidefrom said human; and (b) correlating said mutations in saidpolynucleotide or polypeptide from said human with a autism or apropensity for autism.
 2. The method as claimed in claim 1, wherein saidpolynucleotide or polypeptide obtained from said human is obtained byamplifying a polynucleotide, in its wild-type (nonmutated) form, of SEQID NO: 12, amplifying a fragment of said polynucleotide or amplifying amessenger RNA of said polynucleotide.
 3. The method as claimed in claim1, wherein said polynucleotide or polypeptide obtained from said humanis obtained by amplifying a polynucleotide encoding, in its wild-type(nonmutated) form, a polypeptide of SEQ ID NO: 14, amplifying a fragmentof said polynucleotide or amplifying a messenger RNA of saidpolynucleotide.
 4. The method as claimed in claim 1, wherein saiddetecting comprises detecting a mutation in the polynucleotide of SEQ IDNO: 12, wherein said mutation results in a mutation in the polypeptideencoded thereby at position
 451. 5. The method as claimed in claim 4,wherein said mutation at position 451 is a substitution of an argininewith a cysteine.
 6. The method as claimed in claim 5, wherein saiddetecting further comprises detecting a mutation in the polynucleotideof SEQ ID NO: 12, wherein said mutation results in a mutation in thepolypeptide encoded thereby at position
 796. 7. The method as claimed inclaim 6, wherein said mutation at position 796 is a substitution of anasparagine with a serine.
 8. The method as claimed in claim 1, whereinsaid detecting comprises detecting a mutation in the polynucleotide ofSEQ ID NO: 12, wherein said mutation results in a mutation in thepolypeptide encoded thereby at position
 796. 9. The method as claimed inclaim 8, wherein said mutation at position 796 is a substitution of anasparagine with a serine.
 10. The method as claimed in claim 1, whereinsaid detecting comprises detecting a mutation in the polypeptide of SEQID NO: 14, wherein said mutation is at position
 451. 11. The method asclaimed in claim 10, wherein said mutation at position 451 is asubstitution of an arginine with a cysteine.
 12. The method as claimedin claim 10, wherein said detecting further comprises detecting amutation in the polypeptide of SEQ ID NO: 14, wherein said mutation isat position
 796. 13. The method as claimed in claim 12, wherein saidmutation at position 796 is a substitution of an asparagine with aserine.
 14. The method as claimed in claim 1, wherein said detectingcomprises detecting a mutation in the polypeptide of SEQ ID NO: 14,wherein said mutation is at position
 796. 15. The method as claimed inclaim 14, wherein said mutation at position 796 is a substitution of anasparagine with a serine.